Experimental designs that take advantage of high-throughput sequencing to generate datasets include RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), sequencing of 16S rRNA gene fragments, metagenomic analysis and selective growth experiments. In each case the underlying data are similar and are composed of counts of sequencing reads mapped to a large number of features in each sample. Despite this underlying similarity, the data analysis methods used for these experimental designs are all different, and do not translate across experiments. Alternative methods have been developed in the physical and geological sciences that treat similar data as compositions. Compositional data analysis methods transform the data to relative abundances with the result that the analyses are more robust and reproducible.

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.

Group I introns are intervening sequences that have invaded tRNA, rRNA and protein coding genes in bacteria and their phages. The ability of group I introns to self-splice from their host transcripts, by acting as ribozymes, potentially renders their insertion into genes phenotypically neutral. Some group I introns are mobile genetic elements due to encoded homing endonuclease genes that function in DNA-based mobility pathways to promote spread to intronless alleles. Group I introns have a limited distribution among bacteria and the current assumption is that they are benign selfish elements, although some introns and homing endonucleases are a source of genetic novelty as they have been co-opted by host genomes to provide regulatory functions. Questions regarding the origin and maintenance of group I introns among the bacteria and phages are also addressed.

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY. To infect its human host, the bacterial pathogen Staphylococcus aureus must either take up nutrients from the surrounding environment or produce them itself. Previous studies have reported that S. aureus does not produce the amino acids leucine and valine, despite it possessing the genes to do so. In this study, we show that S. aureus does indeed produce leucine and valine, but only under certain nutritional conditions. We select for mutants of S. aureus able to grow in valine-depleted environments to uncover genetic variants that enable valine production. We discover genetic variants in a repressor protein and a region of non-coding DNA that both, when properly functioning, inhibit production of leucine and valine under nutrient-rich conditions. We further identify the nutritional conditions where the inhibition of leucine and valine production is relieved, thus revealing a previously overlooked role for another amino acid, isoleucine, in influencing nutrient metabolism. We show that isoleucine levels also influence expression of genes involved in the ability of S. aureus to cause disease. These findings help to reconcile conflicting reports regarding leucine and valine production in S. aureus and reveal nutritional cues that could influence its ability to cause infection.

Group I and II introns can be considered as molecular parasites that interrupt protein-coding and structural RNA genes in all domains of life. They function as self-splicing ribozymes and thereby limit the phenotypic costs associated with disruption of a host gene while they act as mobile DNA elements to promote their spread within and between genomes. Once considered purely selfish DNA elements, they now seem, in the light of recent work on the molecular mechanisms regulating bacterial and phage group I and II intron dynamics, to show evidence of co-evolution with their hosts. These previously underappreciated relationships serve the co-evolving entities particularly well in times of environmental stress.